Detection System

Abstract

A method for detecting a target nucleic acid sequence in a sample, by subjecting it to an amplification and taking continuous electrochemical measurements on it during the reaction. The method can be used to determine whet amplification reaction has taken place, to quantitate the amount of target in the sample or to determine sequence characteristics. disclosed is apparatus for use in the method, comprising (i) an amplification reaction vessel which comprises an electrochemic (ii) means for taking continuous electrochemical measurements on a sample contained in the vessel and (iii) tempertature coat measurement means, wherein the electrochemical cell comprises an element formed from an electrically conducting plastics such as a polymer loaded with an electrically conducting material. Further disclosed is a reaction vessel for use in the appar probe for use in the method and a kit for effecting the method.

1 . A method for detecting a target nucleic acid sequence in a sample, the method comprising subjecting the sample to an amplification reaction and taking continuous electrochemical measurements on the sample in situ during the amplification reaction, wherein the amplification reaction is conducted in the presence of an oligonucleotide probe that is specific for a region of the target nucleic acid sequence and includes an electrochemical label, which allows binding of the probe to the target sequence to be electrochemically monitored wherein a change in electrochemical measurement indicates the presence of the target in the sample. 
   
   
       2 . The method according to  claim 1 , wherein the electrochemical label comprises a modified base residue which has different electrochemical properties to native bases in the target sequence and otherwise present in the amplification reaction. 
   
   
       3 . The method according to  claim 2 , wherein the modified base residue is inosine, xanthosine, hypoxanthine, xanthine, 1-methyladenine, 6-methyladenine, 6-benzyladenine, 8-oxyadenine or 2-aminopurine or a base which is modified by osmium tetroxide, pyridine or chloroacetaldehyde. 
   
   
       4 . The method according to  claim 1 , wherein the probe comprises a pair of labels which will undergo a detectable redox reaction when in close proximity to each other. 
   
   
       5 . The method according to  claim 4 , wherein the probe comprises an oligonucleotide having complementary 5′ and 3′ sequences optionally contiguous with an amplification primer, a probe which is hydrolysed during the amplification reaction, a two-part probe the parts of which hybridise in close proximity to each other on the target sequence, or a probe having first and second labels separated by a region which forms a site for a restriction enzyme when in double stranded form. 
   
   
       6 . The method according to  claim 1 , wherein the probe is designed such that it can be released intact from the target sequence and so may take part again in the reaction. 
   
   
       7 . The method according to  claim 1 , wherein the probe is free in solution. 
   
   
       8 . The method according to  claim 1 , wherein the probe is specific either for a splice region of RNA or an intron in DNA, so that only one of the amplified RNA or amplified DNA is detected. 
   
   
       9 . The method according to  claim 1 , wherein the amplification reaction is effected in the presence of a DNA binding agent which facilitates electrochemical measurement. 
   
   
       10 . The method according to  claim 9 , wherein the DNA binding agent comprises an intercalating dye such as ethidium bromide, SybrGold, SybrGreen, PicoGreen, or acridine orange, a single stranded binding protein such as  E. coli  SSB, cisplatin or an electrochemical dye such as Hoechst 33258. 
   
   
       11 . The method according to  claim 9 , wherein the electrochemical measurement is a result of interaction between the probe and the DNA binding agent. 
   
   
       12 . The method according to  claim 1 , further comprising determining a particular reaction condition, characteristic of said sequence, at which the electrochemical measurements change as a result of destabilization or formation of a duplex in the amplification reaction wherein the change in electrochemical measurement is determinative of the characteristic of the target sequence. 
   
   
       13 . (canceled) 
   
   
       14 . The method according to  claim 1 , wherein the target sequence is an internal control sequence. 
   
   
       15 . The method according to  claim 1 , wherein the electrochemical measurements are temperature dependent electrochemical measurements. 
   
   
       16 . The method according to  claim 1 , wherein the electrochemical measurements are potentiometric, conductometric or amperometric, coulometric, and/or voltametric or polarographic measurements. 
   
   
       17 . The method according to  claim 1 , wherein the electrochemical measurements are used to determine whether and/or to what extent an amplification reaction has taken place. 
   
   
       18 . The method according to  claim 1 , wherein the electrochemical measurements are used to quantitate the amount of the target nucleic acid sequence in the sample. 
   
   
       19 . The method according to  claim 1 , wherein the electrochemical measurements are temperature dependent electrochemical measurements which allow characterization of amplification species. 
   
   
       20 - 22 . (canceled) 
   
   
       23 . A probe for use in a method for detecting a target nucleic acid sequence in a sample, wherein the probe is specific for a region of the target nucleic acid sequence and includes an electrochemical label, which allows binding of the probe to the target sequence to be electrochemically monitored, and the probe is free in solution. 
   
   
       24 . An apparatus for use in a method for detecting a target nucleic acid sequence in a sample, the apparatus comprising (i) an amplification reaction vessel which comprises an electrochemical cell, (ii) means for taking continuous electrochemical measurements on a sample contained in the amplification reaction vessel and (iii) temperature control and measurement means, wherein the electrochemical cell comprises at least one element formed from an electrically conducting plastics material. 
   
   
       25 - 45 . (canceled)